JPH0657694B2 - Novel nitroxyl compound, production method thereof and NMR diagnostic agent containing the same - Google Patents

Abstract

Diagnostic media suitable for NMR diagnoses contain compounds of Formula I <IMAGE> (I) wherein is a single bond or a double bond, X is the grouping -(CH2)n- or, if is a single bond, also the grouping -NHCO(CH2)n- wherein n means 0 to 4, m means the numbers 0, 1, or 2, R1 is an alkyl residue substituted by hydroxy groups, acyloxy groups and/or alkylidenedioxy groups, R2 has the same meanings as R1 or is a hydrogen atom or an alkyl residue, R3 and R4 are alkyl residues, and R5 and R6 are alkyl residues optionally substituted by hydroxy groups.

Description

TECHNICAL FIELD The present invention relates to a novel nitroxyl compound described in the claims, a process for producing the same, and an NMR diagnostic agent containing the same.

Problem to be Solved by the Invention An object of the present invention was to find a compound suitable for producing a diagnostic agent used in NMR diagnosis.

Means for Solving the Problems (I) This problem is according to the invention of the general formula I: [Wherein B is a protein, sugar or lipid residue or group Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R
₃ and R ₄ represent an alkyl group, and R ₅ and R ₆ represent an alkyl group optionally substituted by a hydroxy group].

Compounds of general formula I are suitable as substituents B for example protein groups such as albumin, immunoglobulins or monoclonal antibodies, or sugar groups such as glucose or glucose derivatives or for example dipalmitoylphosphatidyl-ethanolamine. Has a lipid group or a group —NR ₁ R ₂ . In the formula, R ₁ represents an alkyl group which is substituted by a hydroxy group, an acyloxy group or an alkylidenedioxy group and which has in particular 2 to 8, in particular 2 to 6 carbon atoms. All the carbon atoms of this alkyl group may have a hydroxy group except for the C atom which is bonded to the amide nitrogen. A suitable substituent R ₁ is for example:
2-hydroxyethyl group, 2-hydroxypropyl group,
2-hydroxy-1-methyl-ethyl group, 3-hydroxypropyl group, 2,3-dihydroxypropyl group, 2-
Hydroxy-1- (hydroxymethyl) -ethyl group, 2
-Hydroxybutyl group, 2,3-dihydroxybutyl group, 2,3,4-trihydroxybutyl group, 2,3-dihydroxy-1-hydroxymethylpropyl group, 2,
3,4,5-tetrahydroxypentyl group or 2,
It is a 3,4,5,6-pentahydroxyhexyl group.
The hydroxy groups may be present in free form, esterified or ketalized form. Suitable esters are preferably from 2 to 2, such as acetic acid, propionic acid, butyric acid, isobutyric acid, trimethylacetic acid, valeric acid or caproic acid.
As derived from alkanecarboxylic acids having 6 carbon atoms. The ketalized hydroxy groups are especially acetonides.

The substituent R ₂ may represent the same as the substituent R ₁ , but
Preferably a hydrogen atom or an alkyl group having up to 4 carbon atoms such as ethyl, propyl, isopropyl,
Or in particular it represents a methyl group.

The substituents R ₃ and R ₄ represent lower alkyl radicals having 1 to 4 carbon atoms such as ethyl, propyl, isopropyl and in particular methyl radicals.

The substituents R ₅ and R ₆ preferably represent the same as R ₃ and R ₄ . However, this is for example a hydroxymethyl group, 1-
Hydroxyethyl group, 2-hydroxyethyl group, 1,2
-Dihydroxyethyl group, 1-hydroxypropyl group,
It may represent an alkyl group having up to 4 carbon atoms, substituted by a hydroxy group, such as a 1,2-dihydroxypropyl group or a 1,2,3-trihydroxypropyl group.

In the group (CH ₂ ) _n , n preferably represents 0 or 1;
In --NHCO (CH ₂ ) _n , n preferably represents 3 or 4 and especially 2.

Among the compounds represented by the general formula I, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (2-hydroxyethyl)-or-(2,3-
Dihydroxypropyl) -amide is a known substance (see "Tetrahedron" Vol. 33, 1977, page 2969).

By the way, it has been surprisingly found that these substances and the other unknown compounds of Claim 1 are remarkably suitable for the production of diagnostic agents used in NMR diagnosis. Was done. There is a need for NMR diagnostic agents that are particularly well-tolerated but stable, well-dissolved and of sufficient selectivity for a wide variety of purposes. Claims No. 1
The novel compound described in the section is m, X, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ represent the above, Is a double bond, m and n are zero, R ₃ , R ₄ ,
R ₅ and R ₆ each represent a methyl group, and when R ₂ is a hydrogen atom, R ₁ is 2,3-dihydroxy-prop-2.
-Yl group, 2,3,4-trihydroxybutyl group, 1,
3,4-trihydroxybut-2-yl group, trishydroxymethylmethane group, 2,3,4,5-tetrahydroxypentyl group, 1,2,4,5-tetrahydroxypent-3-yl group, 2 , 3,4,5,6-Pentahydroxyhexyl group or 1,3,4,5,6-pentahydroxyhex-2-yl group. These novel compounds of the general formula I ′, in particular those in which the hydroxy groups are not present in esterified or ketalized form, fulfill this requirement. For example, the compounds of general formula I linked to proteins are particularly suitable for the diagnosis of tumors and infarctions. For the examination of the liver and spleen, for example complex lipids or inclusion compounds with liposomes, which are present, for example, as single or multilamellar phosphatidylcholine-cholesterol-vesicles, are particularly suitable.

The diagnostic agent according to the present invention can be applied in the intestine or outside the intestine, especially in the vein, the artery or the lumbar spine.

Means for Solving the Problems (II) The novel compound represented by the general formula I ′ can be produced by a method known per se by the method according to claims 25 to 28.

The preparation of the novel nitroxyl of the general formula I ′ from the compound of the general formula II in which B represents a —NR ₁ R ₂ — group is carried out under conditions known to those skilled in the art. There, the compound of general formula II is oxidized with, for example, hydrogen peroxide in the presence of a tungstate catalyst such as sodium tungstate.
Oxidation is performed particularly well with organic peracids such as perbenzoic acid, 3-chloroperbenzoic acid and peracetic acid.

The formation of amides from the corresponding carboxylic acids and amines likewise takes place in a conventional manner (“Houben-Weyl, Methoden de
r Organischen Chemie ”Volume 15/2, 1974, first
See page). For example, the carboxylic acid can be converted to the corresponding acid chloride or mixed anhydride (for example with trifluoroacetic anhydride or methyl chloroformate), which can be reacted with an amine. Furthermore, both components can be condensed in the presence of a dehydrating agent (eg N, N'-carbonyldiimidazole or dicyclohexylcarbodiimide).

The optional subsequent removal of the protecting groups is likewise carried out in a conventional manner. Ketal elimination with acids and ester elimination with alcoholic acids or bases are mentioned.

The formation of a complex with a biomolecule is also carried out by a method known per se, for example, with a nucleophilic group of a biomolecule such as an amino group, a phenol group, a sulfhydryl group or an imidazole group and an activated derivative of the general formula III. The reaction is carried out. Activated derivatives include, for example, salt oxides, mixed anhydrides (eg GEKrejcarek uKL, Tucke
r, "Biochem, Biophys.Res.Commun", 1977, 5
(See page 81), activated esters, nitrenes or isothiocyanates.

The starting compounds of the process according to the invention are either known or can be prepared by methods known per se. It has been found that the synthesis of known compounds can often be carried out in a significantly simpler way than that described in the literature, as the preparation examples below show.

2,2,5,5-Tetramethyl-pyrroline-3-carboxylic acid methyl ester To 1200 m of sodium methylate solution containing 20.69 g (900 mmol) of sodium was added at 10 to 15 ° C. while stirring and cooling. 5-dibromo-2,2
118.19 g (300 mmol) of 6,6-tetramethylpiperidin-4-one hydrobromide are added in portions. After stirring for 30 minutes, evaporate to dryness in vacuo, the residue is taken up in anhydrous diethyl ether, the solid is filtered off, the solution is freshly evaporated to dryness in vacuo and the residue is distilled in a water pump vacuum.

2,2,5,5-Tetramethyl-3-pyrroline-3-carboxylic acid methyl ester distills out as a colorless liquid at 88-89 ° C / 15 mmHg. 54.2g (91% of theory)
Is obtained.

The compound has a purity of 92.3% according to a gas chromatogram,
It is pure enough for further processing.

2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid methyl ester dichloromethane 7
2.38 g (73 mmol) of 2,2,5,5-tetramethyl-3-pyrroline-3-carboxylic acid methyl ester was dissolved in 0 m, 0.1 g (1.2 mmol) of sodium acetate was added, and the mixture was heated to -10 ° C. While cooling and stirring, additionally contain 0.8 g (9.6 mmol) of sodium acetate, 37
A cooled solution of 30% (146 mmol)% peracetic acid is added in portions. After the addition is complete, stir for 4 hours. It is then diluted with dichloromethane and stirred into sodium bicarbonate solution to neutralize the acetic acid. The organic phase is separated off, dried over sodium sulphate and evaporated to dryness in vacuo. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid methyl ester crystallizes from diethyl ether into needle crystals having a melting point of 87 to 89 ° C.

The yield is 12.63 g (87.3% of theory).

2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid 2,2,40 in 40m distilled water and 10m ethanol
5,5-tetramethyl-3-pyrroline-1-oxyl-
To a solution of 15.10 g (76.17 mmol) of methyl 3-carboxylic acid 4.40 g of sodium hydroxide in 40 m of distilled water.
57 g (114.26 mmol) were added. Heat to 60 ° C. with stirring for 1.5 hours, dilute with 200 m of water, acidify to a pH value of 3 with 4N sulfuric acid and extract the acid with methylene chloride. The organic phase is dried over sodium sulphate and evaporated to dryness in a vacuum. 2,2,5,5-tetramethyl-3-pyrroline-1
-Oxyl-3-carboxylic acid crystallizes from ethanol / diethyl ether (mp 221-223 ° C). The yield is 13.82 g (98.5% of theory).

2,2,5,5-Tetramethylpyrrolidine-3-carboxylic acid methyl ester.

87.80 g (479 mmol) of 2,2,5,5-tetramethyl-3-pyrroline-3-carboxylic acid methyl ester was dissolved in 1000 m of methanol to give Raney Nickel 1
Hydrogenation is carried out in the presence of 1 g at a starting pressure of 180 bar at room temperature. The catalyst is filtered off with suction, the solution is treated with activated carbon, concentrated in vacuo and the residue is distilled in a water pump vacuum.
2,2,5,5-Tetramethylpyrrolidine-3-carboxylic acid-methyl ester is distilled at 86 to 87 ° C./14 mmHg. 77.1 g (88% of theory) are obtained. The compound has a purity of 98.7% according to a gas chromatogram.

_{C 10 H 19 NO 2 (185.27} ) Calculated: 64.83C 10.34H 7.56N Found: 64.71C 10.44H 7.44N 2,2,5,5- tetramethyl-1-oxyl-3-carboxylic acid methyl ester.

2,2,5,5-Tetramethylpyrrolidine-3-carboxylic acid methyl ester (9
8.7%) To a solution of 20.30 g (108.15 mmol) 0.14 g (1.6 mmol) sodium acetate is added and the solution is cooled to -10 ° C. To the stirred solution was added peracetic acid (37
%, Sulfuric acid 1%) 45.2 g (220 mmol) and sodium acetate 1.22 g (14.7 mmol) suspended in peracetic acid are added dropwise. Stir overnight, neutralize acetic acid with sodium bicarbonate, separate organic layer, dry over sodium sulfate and concentrate in vacuo. The residue is distilled using an oil pump. 20.23 g of an orange oil at 76 ° C / 0.01 torr
(93.4% of theory) is distilled.

_{C 10 H 18 NO 3 (200.26} ) Calculated: 59.98C 9.06H 6.99N Found: 59.89C 9.17H 6.93N 2,2,5,5- tetramethyl-1-oxyl-3-carboxylic acid.

8.75 g (218.87) sodium hydroxide in 120 m of distilled water
(2 mmol), 2,2 in 20 m ethanol
5,5-tetramethylpyrrolidine-1-oxyl-3-
Carboxylic acid methyl ester 29.22 g (145.91 mmol)
Solution is added. Heat at 60 ° C. for 90 minutes with stirring, dilute with 200 m of water and acidify to a pH value of 3 with 4N sulfuric acid. The acid is taken up in dichloromethane, the organic solution is dried over sodium sulphate and evaporated to dryness in a vacuum. The residue,
Crystallize from dichloromethane / diethyl ether. Thus, 2,2,5,5-tetramethyl-pyrrolidine-1-oxyl-3-carboxylic acid having a melting point of 197 to 199 ° C.
26.12 g (96.1% of theory) are obtained.

_{C 9 H 16 NO 3 (186.23} ) Calculated: 58.05C 8.66H 7.52N Found: 58.11C 8.72H 7.35N succinate - (2,2,6,6-tetramethyl-1-oxyl-4-piperidyl) -Monoamide.

55 g (= 321 mmol) of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl are dissolved in 55 m of pyridine. While stirring, 40.7 g (407%) of succinic anhydride in 500 m of tetrahydrofuran was added.
Mmol) is added dropwise. After stirring for 12 hours at room temperature, evaporate to dryness in vacuo. 1000 m of water are added to the residue and the aqueous solution is extracted with ether to remove pyridine. It is then concentrated in vacuo to approx. 200 m, the precipitate formed is stirred in an ice bath for several hours and then suction filtered. After washing with a small amount of ice-cold water and drying in vacuum at 40 ° C., melting point 108-1
81.3g (= theoretical value of 9 as orange crystals at 10 ℃)
3%) is obtained.

_{C 13 H 23 N 2 O 4} (271.34) Calculated: 57.54C 8.54H 10.33N Found: compound according to the invention 57.48C 8.60H 10.41N formula I is a valuable diagnostic agent.
For practical application, it is dissolved or suspended in water or saline and, optionally with the additives customary in galenical formulations, converted into a form suitable for intravascular or oral administration, so that The concentration is in the range of 1 μmol / -1 mol /.

EFFECTS OF THE INVENTION The tolerability of the novel diagnostic agents is clearly superior to that of known comparable compounds. Sodium, potassium, meglumine (Meglumin), which was previously required for salt formation,
e) The toxic effects of other ions are completely or largely eliminated. The osmotic pressure of this concentrated solution of diagnostic agent is significantly reduced. It also significantly improves tolerability after oral and parenteral administration because hypertonic solutions severely damage blood vessels and tissues, affect the heart and circulatory organs, and exert undesired diuretic effects. Because it does.

The compounds according to the invention have a favorable effect on the relaxation times of the protons. It has better bioavailability compared to the known nitroxyl free radicals and is significantly less toxic than this, as the following tests show: Mice weighing 18-22 g receive different amounts of substance. 1 kg
A 0.5 mM aqueous solution (adjusted to pH 7-7.5) is injected intravenously into the tail vein. After 7 days, the survival rate of the mouse is confirmed, and from this, the LD ₅₀ is confirmed in the usual manner in mmol / kg animal.

The following table shows the results obtained in this case, known compounds 1 and 2 (“J.Compt.Assist.Tomogr.”, Volume 7 (1983).
Year) Page 184).

In essence, this has led to the successful production of new diagnostic agents, which open up new possibilities in diagnostic medicine. With the development of new types of imaging methods, especially in diagnostics, this seems highly desirable and necessary.

The following relaxivity was measured [[relaxation times T ₁ and T ₂ are measured by
In mini-spec _{(Minispec) p 20 (Bruker)} , 0.46 Tesla (Tesla) (= 20MHz), measured at 39 ℃], the next LD
₅₀ -value was measured: The accompanying photographs show examples for in vivo NMR-diagnosis. Substances used: 2,2,5,5-tetramethylpyrrolidine-1-oxyl-carboxylic acid- (1,3,4-
Trihydroxybut-2-yl) -amide (Example 24).

Test animal: Colon cancer HT29 (10 ⁷ cells applied intraperitoneally: after about 1 week the tumor grows to the size shown in the picture)
(Nacktmaus: female about 25 g).

Measurement parameters: The animals were examined on a General Electric nuclear spin tomography machine with a magnetic 2 Tesla. Before and after contrast agent application, _{T 1} - wt spin - echo sequence _{[T 1 -gewichteten Spin-Echo-} Sequ
ez: Echo time: T _E = 15 seconds, recall: T _R = 400
m sec; Layer thickness: 3 mm in axial direction; Image size (Field of view =
FOV): 70 × 70 mm] was used to image the inside of a predetermined region (tumor, kidney). Concentration of reference solution: 0.1 mol /, dose: 1 mmol / kg.

Conduct: Nembutal (Nembutal: 0.9% NaC)
Anesthesia at 1: 4 dilution (apply 100 μ subcutaneously). Subsequently, a cannula was placed in the tail vein and fixed.
The mouse was then tomographically imaged and double applied (after measurement of the null value image). That is, the contrast agent is 1 mmol /
kg (intravenous) was applied. Apply a bolus,
Then a second dose of 1 mmol / kg was infused over 5 minutes.

FIG. 1 shows a null image of a given area (ie no substance has been applied yet). On the right side is a tumor, two large blood vessels (black circles) and a fat-enclosed intestine in the left area. Fat is recognizable due to its high signal strength. This animal is hungry. The spot on the left is a reference material that helps to understand the true signal strength difference within the tissue.

Fig. 2: Double application was performed after null value photography, and measurement was performed immediately after the application was completed. The tumor appears darker on this picture, which means that the substance has already been distributed in the vascular circulation and has a very high signal intensity, which is accompanied by a total intensity of the image being too high. Because it is lowered so that it does not become a modulation. It is clearly seen that this substance is present in the connective tissue capsule of the tumor (very strongly blood flow through).

Fig. 3: Measurement was performed again after 30 minutes from the same viewpoint without moving the mouse. The tumor has again reached its original gray color. This means that this substance is gradually eliminated from the blood circulation system. This is evident from the appearance of the two kidneys in the upper region. This kidney filters the blood, so that the substance is present in the kidney tissue and is subsequently excreted via urine.

FIG. 4: All three photographs are shown again for better observation of the time course.

Upper left: null value, upper right: immediately after injection (subcutaneous), lower left: 30 minutes after subcutaneous injection The present invention will be described in detail with reference to the following examples.

Examples Example 1 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- [2-hydroxy-2- (2,2
-Dimethyl-1,3-dioxolan-4-yl) -ethyl] -amide 2,2,5,5 in 200 m anhydrous tetrahydrofuran
8.03 g of triethylamine in a solution of 14.82 g (79.58 mmil) of tetramethyl-pyrrolidine-1-oxyl-3-carboxylic acid while stirring, cooling and covering with argon.
(79.58 mmol) is added. After cooling to -5 °, 4
In the course of 5 minutes, 8.638 g (79.59 mmol) of chloroformic acid ethyl ester in 10 m of anhydrous tetrahydrofuran are added dropwise with stirring. Stir for 30 minutes at -5 ° C. Then 2-
Amino-1- (2,2-dimethyl-1,3-dioxolan-4-yl) -ethanol 12.83 g (79.59 mmol)
Is added separately. After 30 minutes the cooling was removed and 3 at room temperature.
Stir for hours. Thereafter, it is diluted with 200 m of anhydrous ether, the solid is suction filtered and washed with anhydrous ether. The organic solution is evaporated to dryness in a vacuum. The residue is taken up in dichloromethane and washed with semisaturated sodium bicarbonate solution,
Dry over sodium sulfate and evaporate to dryness in vacuo. The yellow oil which remains crystallizes after addition of diethyl ether. 2,2,5,5-tetramethylpyrrolidine-1-
Oxyl-3-carboxylic acid- [2-hydroxy-2-
(2,2-Dimethyl-1,3-dioxolan-4-yl) -ethyl] -amide was recrystallized from ester acetate and melted at 144 ° C. to give 14.79 g of the compound (theoretical value of 56.4).
%) Is obtained. 4.0 g of the starting acid is recovered from the bicarbonate solution.

Example 2 2,2,5,5-tetramethylpyrrolidine-1-oxy
Le-3-carboxylic acid- (2,3,4-trihydroxybutane
15) water containing 0.1 m (3.6 mmol) of concentrated sulfuric acid.
2,2,5,5-tetramethylpyrrolidine in 0 m
-1-oxyl-3-carboxylic acid- [2-hydroxy-
2- (2,2-dimethyl-1,3-dioxolane-4-
Ill) -ethyl] -amide 13.9 g (42.46 mol) suspended
Let Heat to 40-50 ° for 3 hours with stirring
It Cool the solution and add sulfuric acid to the amber light ion exchanger
(Amberlite) IRA410OH^--Neutralize in form. Exchanger
Is filtered off and washed with water. The combined solution is evaporated to dryness in a vacuum.
To dry up. 2,2,5,5-tetramethylpyrrolidine-1
-Oxyl-3 (2,3,4-trihydroxybutyl)
The amide is crystallized from ethanol, yellowish with a melting point of 172 ° C.
10.97 g (89.3% of theory) of colored crystals are obtained. From water
Recrystallized to give yellow needle crystals with a melting point of 189-91 ° C.
can get.

Example 3 2,2,5,5-Tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- [N- (2,3,4,5,6-pentahydroxyhexyl) -N-methyl] -amide dimethylformamide 2,2,5,5-in 300m
To a solution of 19.93 g (107 mmol) of tetramethyl-pyrrolidine-1-oxyl-3-carboxylic acid, 12.05 g of triethylamine while stirring, cooling and covering with argon.
(119 mmol) is added. After cooling to -10 ° C, 3
In 0 minutes, 12.92 g (119 mmol) of ethyl chloroformate was dissolved in 30 m of anhydrous tetrahydrofuran and added. After 10 minutes, 10.89 g of N-methylglucamine (1
(07 mmol) are added in portions. Stir at 0 ° C. for 2 hours, remove the cooling and stir overnight at room temperature. The solid is filtered off and washed with dimethylformamide and anhydrous tetrahydrofuran.

The combined solution is concentrated in vacuo and finally with an oil pump. The rest
Take the residue in anhydrous tetrahydrofuran and use diethyl ether.
Re-sink from. Dissolve the precipitated oil in water and ionize.
Amberlite (Amberlite IR120H⁰Shape and IRA410OH⁰Shape)
It is obtained without any ions by reasoning. Vacuum the yellow solution
Evaporate to dryness in. The residual syrupy material has a strong moisture absorption.
Store in vacuo over phosphorous pentoxide as it is volatile. Two
2,5,5-tetramethylpyrrolidine-1-oxyl-
Carboxylic acid- [N- (2,3,4,5,6-pentahydr
Roxyhexyl) -N-methyl] -amide 24.8 g (theory)
63.8% of the value) as a yellow to orange syrup
To be

C₁₆H₃₁N₂O₇Molecular weight (363.43) Calculated: 52.88C 8.60H 7.71N Found: 52.61C 8.78H 7.59N Example 4 2,2,5,5-tetramethylpyrrolidine-1-oxy
Le-3-carboxylic acid- (2,3-dihydroxypropyi
) -Amidodichloromethane 2,2,5,5-tetradichloromethane in 150 m
Methylpyrrolidine-1-oxyl-3-carboxylic acid 13.6
To 1 g (73.1 mmol) of solution, stir, cool and stir.
8.13 g (80.3 mm) of triethylamine while covering with gon
Add). After cooling to -10 ° C, dichloromethane
8.72 g (80.3 mi) of chloroformic acid ethyl ester in 10 m
Limol) was added dropwise, and the mixture was stirred at -5 ° C for 20 minutes and then dimethyl
2,3-dihydroxypro in 40m of luacetamide
6.66 g (73.1 mmol) of pyramine are added dropwise. Cooling
Remove, stir after 1 hour and filter the solids with suction,
After washing with anhydrous tetrahydrofuran, in vacuum and finally
Evaporate to dryness with an oil pump. Extract the residue with anhydrous ether
Then, it is dissolved in water and the ion exchanger (Amberlite IR120H⁺
Shape and IRAOH^-Form) to obtain without ions
It The yellow solution was evaporated to dryness in vacuo, 2,2,5,5-
Tetramethylpyrrolidine-1-oxyl-3-carvone
Acid- (2,3-dihydroxypropyl) -amide 12.4g
(65.5% of theoretical value) is yellow to orange syrupy
Obtained as.

C₁₂H_{twenty three}N₂O_Four Molecular weight 259.33 Calculated: 55.58C 8.94H 10.80N Measured: 55.33C 9.19H 10.62N Example 5 2,2,5,5-tetramethylpyrrolidine-1-oxy
Le-3-carboxylic acid-bis (2-hydroxyethyl)-
Amide 2,2,5,5 in anhydrous tetrahydrofuran 150m
-Tetramethyl-pyrrolidine-1-oxyl-3-cal
Stir and cool to a solution of 12.91 g (69.32 mmol) of boric acid.
And while covering with argon, triethylamine 7.69g
(76.0 mmol) is added. After cooling to -10 ° C,
Ethyl chloroformate in 10m of water tetrahydrofuran
Stell 8.25 g (76.0 mmol) was added dropwise, and at -5 ° C, 20
After stirring for a minute, diethanol
7.2 g (69.32 mmol) of min are added dropwise. Remove cooling
Then, stir after 1 hour. Next, anhydrous ether 200m
Dilute with and filter the solid with suction, which is post-extracted with ether.
Wash and concentrate in vacuo. Take the residue in water and ion exchange.
Amberlite (Amberlite IR120H⁺Shape and IRA410OH^-Shape)
It is obtained without any ions by reasoning. Yellow neutral solution
Was evaporated to dryness in vacuo to give 2,2,5,5-tetramethyl
Pyrrolidine-1-oxyl-3-carboxylic acid-bis (2
-Hydroxyethyl) -amide 11.64 g (theoretical 61.4
%) As a yellowish to orange syrup.
It

C₁₃H_{twenty five}N₂O_Four Molecular weight 273.36 Calculated: 57.12C 9.22H 10.25N Measured: 57.01C 9.38H 10.10N Example 6 2,2,5,5-tetramethylpyrrolidine-1-oxy
-3-carboxylic acid- (1,3-dihydroxyprop-
2-yl) -amido 2,2,5,5 in 200 m of anhydrous tetrahydrofuran
-Tetramethylpyrrolidine-1-oxyl-3-carb
11.41 g (61.27 mmol) of acid and triethylamine
A solution consisting of 6.20 g (61.27 mmol) was stirred at -5 ° C.
Tetrahydrofuran 10 while stirring and covering with argon
6.65 g (61.27 mm) of ethyl chloroformate in m
Mol) is added dropwise. After 30 minutes, powdered 2-amino
-1,3-propanediol 5.59 g (61.3 mmol)
Add separately. Remove the cooling bath and stir after 5 hours. So
After that, dilute with anhydrous diethyl ether 200m and solid
Filtered off with suction, washed with ether and washed in vacuo.
Concentrate. The residue is taken up in water and the ion exchanger (Amberlite
IR120H⁺Shape and IRA410OH^-Shape)
Obtained without on. Evaporate the neutral yellow solution in vacuo to dryness
2,2,5,5-tetramethylpyrrolidine-1-
Oxyl-3-carboxylic acid- (1,3-dihydroxypropyl
Lop-2-yl) -amide 10.1 g (64.4% of theory)
Is obtained as a dark yellow syrap.

C ₁₂ H ₂₃ N ₂ O ₄ (259.33) Calculated: 55.58C 8.94H 10.80N Found: 55.37C 9.19H 10.64N Reference Example 1 (preparation of starting material of Example 6) 2,2,5,5-tetra Methyl-3-pyrroline-3-carboxylic acid- (2,2-dimethyl-1,3-dioxolan-4-ylmethyl) -amide (2,2-dimethyl- in 700 m isopropanol)
1,3-dioxolan-4-yl) -methylamine 22.9
A solution consisting of 3 g (175 mmol) and 58.9 g (525 mmol) potassium-t-butyrate was stirred,
While cooling to −5 to −0 ° C. and blanketing with argon,
68.94 g (175 mmol) of 5-dibromo-2,2,6,6-tetramethyl-piperidin-4-one hydrobromide are added in portions. A white mud forms. After stirring for 1 hour without cooling, the solid is filtered off with suction and evaporated to dryness in vacuo. The residue is partitioned between dichloromethane and water. The organic phase is dried over sodium sulphate and evaporated to dryness in a vacuum. The residue crystallizes. After recrystallization from pentane, 2,2,5,5-tetramethyl-3- having a melting point of 55 to 57 ° C.
Pyroline-3-carboxylic acid- (2,2-dimethyl-1,
3-dioxolan-4-ylmethyl) -amide 30.1 g
(60.9% of theory) is obtained.

C ₁₅ H ₂₆ N ₂ O ₃ (282.40) Calculated value: 63.35C 9.92H 9.82N Measured value: 63.11C 9.72H 9.99N Example 7 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-carboxylic acid- (2,2-dimethyl-1,3
-Dioxolan-4-ylmethyl) -amide 2,2,5,5-in 200m anhydrous diethyl ether
Tetramethyl-3-pyrroline-3-carboxylic acid- (2,
2-Dimethyl-1,3-dioxolan-4-ylmethyl) -amide 15.88 g (56.23 mmol) was dissolved and -5
Cool to 0 ° C. and add a solution of 23.92 g (112.4 mmol) of 3-chloroperbenzoic acid (80%) in 150 ml of anhydrous ether in portions with stirring. The cooling bath is removed, stirred after 2 hours, stirred in 200 m of a soda solution containing 29 g of sodium carbonate decahydrate and the product is taken up in diethyl ether. The ether solution is dried over sodium sulphate and evaporated to dryness in vacuo. Thus, 2, 2,
5,5-tetramethyl-3-pyrroline-1-oxyl-
16.46 g of 3-carboxylic acid- (2,2-dimethyl-1,3-dioxolan-4-ylmethyl) -amide (9 theoretical)
8.4%) is obtained as an orange syrupy product.

C₁₅H_{twenty five}N₂O_Four (297.38) Calculated value: 60.59C 8.47H 9.42N Measured value: 60.33C 8.68H 9.30N Example 8 2,2,5,5-tetramethyl-3-pyrroline-1-o
Xyl-3-carboxylic acid- (2,3-dihydroxy pro
Pill) -amide 2,2,5,5-tetramethyl-3-pyrroline-1-o
Xyl-3-carboxylic acid- (2,2-dimethyl-1,3
-Dioxolan-4-ylmethyl) -amide 14.2 g (4
Concentrated sulfuric acid 0.1 m (3.6 mmol) contains 7.8 mmol)
Suspended in 100m of distilled water and stored at 50 ° C for 3
Stir for hours. After cooling, the ion exchanger (Amberlite
IRA410OH^-Form) to neutralize. Wash the resin with water and combine
The concentrated solution is concentrated in vacuo. Use the oil pump to remove the residue from the chamber.
Dry on warm. Thus, it has a melting point of 131-133 ° C.
Crystalline 2,2,5,5-tetramethyl-3-pyrrolid
-1-oxyl-3-carboxylic acid- (2,3-dihydride
Roxypropyl) -amide 10.48 g (85.3% of theory)
Is obtained.

C ₁₂ H ₂₁ N ₂ O ₄ (257.31) Calculated: 56.02C 8.23H 10.89N Found: 55.87C 8.39H 10.74N Reference Example 2 (preparation of starting material of Example 9) 2,2,5,5-tetra Methyl-3-pyrroline-3-carboxylic acid- [N- (2,2-dimethyl-1,3-dioxolan-4-ylmethyl) -N-methyl] -amide As in Reference Example 1, 3,5-dibromo -2, 2, 6, 6
-Tetramethylpiperidin-4-one hydrobromide 68.9
4 g (175 mmol) and potassium t-butyrate 5
8 g (525 mmol) and N-methyl-N- (2,
25.41 g (115 mmol) of 2-dimethyl-1,3-dioxolan-4-ylmethyl) -amine are reacted in 600 m of isopropanol.

2,2,5,5-tetramethyl-3-pyrroline-1-3
-39.43 g (76% of theory) of carboxylic acid- [N- (2,2-dimethyl-1,3-dioxolan-4-yl-methyl) -N-methyl] -amide are orange syrupy products. Obtained as.

_{C 10 H 28 N 2 O 3} (296.41) Calculated: 64.84C 9.52H 9.45N Found: 64.59C 9.73H 9.27N Example 9 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl - 3-carboxylic acid- [N- (2,2-dimethyl-
1,3-Dioxolan-4-ylmethyl) -N-methyl] -amide Similar to Example 7, 2,2,5,5-tetramethyl-3-pyrroline-3-carboxylic acid-N-[(2,2 -Dimethyl-
1,3-Dioxolan-4-yl) -methyl] -N-methylamide 14.97 g (50.50 mmol) are reacted with 2-chloroperbenzoic acid (80%) 22.0 g (101 mmol) in 300 ml of anhydrous diethyl ether. . Thus
Shows a melting point of 69 to 71 ° C. after crystallization from acetic ester,
2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- [N- (2,2-dimethyl-
13.12 g (83.5% of theory) of 1,3-dioxolan-4-ylmethyl) -N-methyl] -amide are obtained.

C ₁₆ H ₂₈ N ₂ O ₃ (296.41) Calculated: 61.51C 9.03H 8.97H Found: 61.80C 8.98H 8.87H Example 10 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-Carboxylic acid-N- (2,3-dihydroxypropyl) -N-methyl-amide 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid as in Example 8. -[N- (2
2-Dimethyl-1,3-dioxolan-4-ylmethyl) -N-methyl] -amide 11.49 g (36.9 mmol)
Is saponified in 100 m of distilled water containing 0.05 m (1.8 mmol) of concentrated sulfuric acid. So 2, 2, 5, 5
-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid-N- (2,3-dihydroxypropyl) -N
9.18 g (91.7% of theory) of methyl-amide are obtained as an orange syrupy product.

C ₁₃ H ₂₃ N ₂ O ₄ (271.34) Calculated value: 57.55C 8.54H 10.32N Found value: 57.33C 8.75H 10.18N Example 11 2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid- [N -(2,2-Dimethyl-1,3-dioxolan-4-ylmethyl) -N-methyl] -amide 2,2,5,5-tetramethyl-3-pyrroline-3-carboxylic acid- [400m methanol 400 m -(2,2-Dimethyl-1,3-dioxolan-4-yl-methyl]-
To a solution of 22.58 g (76.18 mmol) of amide, 2 g of Raney Nickel (B115Z) are added and hydrogenated at a starting pressure of 180 bar. Absorb exactly 1 mole of hydrogen.
The catalyst is filtered off with suction, the solution is treated with activated carbon and evaporated to dryness in vacuo. Thus, 2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid- [N- (2,2-dimethyl-1,3-dioxolan-4-ylmethyl) -N
21.0 g (92.4% of theory) of methyl-amide are obtained as a pale yellow oil.

_{C 16 H 30 N 2 O 3} (298.43) Calculated: 64.40C 10.13H 9.39N Found: 64.66C 10.21H 9.57N Example 12 2,2,5,5-tetramethyl-1-oxyl-3-carboxylic Acid- [N- (2,2-dimethyl-1,
3-dioxolan-4-ylmethyl) -N-methyl]-
Amide As in Example 7, 2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid-N-[(2,2-dimethyl-1,
3-dioxolan-4-ylmethyl) -N-methyl]-
20.78 g (69.63 mmol) of amide are reacted with 30 g (140 mmol) of 3-chloroperbenzoic acid (80%) in 400 m of anhydrous diethyl ether. 2, 2, 5, 5
-Tetramethyl-pyrrolidine-1-oxyl-3-carboxylic acid-N-[(2,2-dimethyl-1,3-dioxolan-4-ylmethyl) -N-methyl] -amide 17.52
g (80.3% of theory) are obtained as an orange syrupy.

C ₁₆ H ₂₉ N ₂ O ₄ (313.42) Calculated: 61.32C 9.33H 8.94N Found: 61.61C 9.40H 8.83N Example 15 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic Acid-N- (2,3-dihydroxypropyl) -N-methyl-amide As in Example 9, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- [N- (2 , 2-
Dimethyl-1,3-dioxolan-4-ylmethyl)-
16.60 g (53 mmol) of N-methyl] -amide are saponified in 150 m of distilled water in which 0.05 m (1.8 mmol) of concentrated sulfuric acid is dissolved. Thus 2, 2, 5, 5
13.28 g (91.7% of theory) of tetramethylpyrrolidine-1-oxyl-3-carboxylic acid-N- (2,3-dihydroxypropyl) -N-methylamide are obtained as an orange syrupy product.

C ₁₃ H ₂₅ N ₂ O ₄ (273.36) Calculated: 57.12C 9.22H 10.25N Found: 56.86C 9.39H 10.06N Reference Example 3 (preparation of starting material for Example 14) 2,2,5,5-tetra Methyl-3-pyrroline-3-carboxylic acid- [2-hydroxy-2- (2,2-dimethyl-1,3-dioxolan-4-yl) -ethyl] -amide As in Example 7, 3,5- 51.22 g of dibrom-2,2,6,6-tetramethylpiperidin-4-one hydrobromide
(130 mmol) of potassium t-butyrate 45.05
g (390 mmol) and 2-amino-1- (2,2-
Dimethyl-1,3-dioxolan-4-yl) -ethanol 20.96 g (130 mmol) and isopropanol 50
React at 0 m. Thus, it is crystallized from diethyl ether pentane and exhibits a melting point of 99 to 101 ° C.
2,5,5-Tetramethyl-3-pyrroline-3-carboxylic acid- [2-hydroxy-2- (2,2-dimethyl-
22.93 g (56.5% of theory) of 1,3-dioxolan-4) -yl) -ethyl] -amide are obtained.

C ₁₆ H ₂₈ N ₂ O ₄ (312.41) Calculated value: 61.51C 9.03H 8.97N Measured value: 61.57C 9.33H 8.90N Example 14 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-carboxylic acid- [2-hydroxy-2-
(2,2-Dimethyl-1,3-dioxolan-4) -yl) -ethyl] -amide Similar to Example 7, 2,2,5,5-tetramethyl-3-pyrroline-3-carboxylic acid- [ 2-hydroxy-2-
2.81 g (9 mmol) of (2,2-dimethyl-1,3-dioxolan-4-yl) -ethyl] -amide and 4.72 g (18 mmol) of 3-chloroperbenzoic acid in 100 ml of anhydrous diethyl ether. React. Thus, melting point 1
2,2,5,5-tetramethyl-3- of 04-105 ° C
2.43 g of pyrroline-1-oxyl-3-carboxylic acid- [2-hydroxy-2- (2,2-dimethyl-1,3-dioxolan-4-yl) -ethyl] -amide (theoretical value of 8
3.5%) is obtained.

C₁₆H₂₇N₂O_Five (327.40) Calculated: 58.70C 8.31H 24.43N Measured: 58.46C 9.57H 24.28N Example 15 2,2,5,5-tetramethyl-3-pyrroline-1-o
Xyl-3-carboxylic acid- [2-hydroxy-2-
(2,2-Dimethyl-1,3-dioxolane-4-i
) -Ethyl] -amide 2,2,5,5-tetramethyl-3-pyrroline-3-ca
Rubonic acid- [2-hydroxy-2- (2,2-dimethyl
-1,3-Dioxolane-4-)-ethyl] -amide 1
Dissolve 5.98 g (51.15 mmol) in 100 m of distilled water
Shitriplex (Titriplex ) III 500 mg (1.
34 mmol), sodium tungstate dihydrate 50
0 mg (1.52 mmol) and 30% hydrogen peroxide 10 m
(88.2 mol) is added. In dark flask at room temperature for 5 days
Stir with, adjust to pH 3 with citric acid and dilute the product.
Extract with methane. Wash the organic solution with water and wash with sodium sulfate.
Dry on um and evaporate to dryness in vacuo. Orange
The oil is taken up in diethyl ether and crystallized. This way
2,2,5,5-tetramer having a melting point of 105 to 106 ° C.
Cyl-3-pyrrolinyl-oxyl-3-carboxylic acid-
[2-hydroxy-2- (2,2-dimethyl-1,3-
Dioxolan-4-yl) -ethyl] -amide 12.79 g
(76.4% of theory) is obtained.

Example 16 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (2,3,4-trihydroxybutyl) -amide As in Example 8, 2,2,5 , 5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- [2-hydroxy-2- (2,2-dimethyl-1,3-dioxolan-4-yl) -ethyl] -amide 15.56 g (47.53 mmol) is saponified in 200 m of distilled water containing 0.15 m (5.4 mmol) of concentrated sulfuric acid. The product crystallizes from water. 2,2,5,5- with a melting point of 183-185 ° C
12.87 g (94.2% of theory) of tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (2,3,4-trihydroxybutyl) -amide are obtained.

_{C 13 H 23 N 2 O 5} (287.34) Calculated: 54.34C 8.07H 9.75N Found: (preparation of starting materials of Example 17) 54.21C 8.28H 9.58N Reference Example 4 2,2,5,5-Tetra Methyl-3-pyrroline-3-carboxylic acid- (5-hydroxy-2,2-dimethyl-1,
3-Dioxepan-6-yl) -amide 3,5-di-bromo-2,2,6, as in Reference Example 1.
6-Tetramethylpiperidin-4-one hydrobromide
78.79 g (200 mmol), potassium t-butyrate 69.34 g and 6-amino-2,2-dimethyl-
35.47 g (220 mmol) of 1,3-dioxepan-5-ol were reacted in 800 m of isopropanol,
Then extract with dichloromethane. Thus, 2,2,5,5-tetramethyl-3-pyrroline-3, crystallized from diethyl ether / hexane and shows a melting point of 125-127 ° C.
-Carboxylic acid- (5-hydroxy-2,2-dimethyl-
1,3-dioxepan-6-yl) -amide 37.66 g
(60.3% of theoretical value) is obtained.

C ₁₆ H ₂₈ N ₂ O ₄ (312.41) Calculated: 61.51C 9.03H 8.97N Found: 61.32C 9.20H 8.83N Example 17 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-carboxylic acid- (5-hydroxy-2,2-
Dimethyl-1,3-dioxepan-6-yl) -amide As in Example 7, 2,2,5,5-tetramethyl-3-pyrroline-3-carboxylic acid- (5-hydroxy-2,2-
Dimethyl-1,3-dioxepan-6-yl) -amide
13.0 g (41.61 mmol) and 3-chloroperbenzoic acid (8
0%) 19.15 g (90.0 mmol) and 1 g of dichloromethane
React in 30 m. Thus, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (5-hydroxy-2,2, which crystallizes from diethyl ether / hexane and exhibits a melting point of 132-133 ° C. −
Dimethyl-1,3-dioxepan-6-yl) -amide
11.23 g (82.5% of theory) are obtained.

C ₁₆ H ₂₇ N ₂ O ₅ (327.40) Calculated value: 58.70C 8.31H 8.56N Measured value: 58.04C 8.59H 8.34N Example 18 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-Carboxylic acid- (1,3,4-trihydroxybut-2-yl) -amide As in Example 8, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic Acid- (5-hydroxy-2,2-dimethyl-1,3-dioxepane-6-
Yl) -amide 12.53 g (38.27 mmol), concentrated sulfuric acid
Saponify in 100 m of distilled water containing 1 m (3.6 mmol). The product crystallizes from a concentrated aqueous solution. Thus, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (1,3,4-trihydroxybut-2-yl) having a melting point of 75 to 77 ° C.
9.60 g (87.3% of theory) of amide are obtained.

C ₁₃ H ₂₃ N ₂ O ₅ (287.34) Calculated: 54.34C 8.07H 9.75N Found: 54.36C 8.32H 9.60N Example 19 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic Acid- (5-hydroxy-2,2-dimethyl-1,3-dioxepan-6-yl) -amide Similar to Example 4, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic Acid 24.23 g (130.11 mmol), triethylamine 13.59 g (133.0 mmol), chloroformic acid ethyl ester 14.87 g (133.0 mmol), 6-amino 2,2-dimethyl-1,3-dioxepan-5-ol 21.44 g (133 mmol) ) Is reacted in 350 m of anhydrous tetrahydrofuran. Crystallized from diethyl ether and showing a melting point of 190-191 ° C., 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (5-hydroxy-2,2-
Dimethyl-1,3-dioxepan-6-yl) -amide
24.83 g (56.7% of theory) are obtained.

C ₁₆ H ₂₉ N ₂ O ₅ (329.42) Calculated: 58.34C 8.87H 8.50N Found: 58.20C 8.99H 8.39N Example 20 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic Acid- (1,3,4-trihydroxybut-2-yl) -amide As in Example 8, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (5-hydroxy 6.0 g (18.21 mmol) of 2,2-dimethyl-1,3-dioxepan-6-yl) -amide was added to 0.05 m of concentrated sulfuric acid.
Saponification is carried out in 100 m of distilled water containing (1.8 mmol). Thus, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (1,
3,4-trihydroxybut-2-yl) -amide 4.83
g (91.7% of theory) are obtained as an orange syrupy product.

C ₁₃ H ₂₅ N ₂ O ₅ (289.35) Calculated: 53.96C 8.71H 9.68N Found: 53.98C 8.91H 9.57N Example 21 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-carboxylic acid- [N- (2,3,4,5,6
-Pentahydroxyhexyl) -N-methyl] -amide Similar to Example 4, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid 12.90 g (70
Mmol), 7.51 g (73.5 mmol) of triethylamine and 8.22 g (73.5 mmol) of ethyl chloroformate were reacted in 150 m of anhydrous tetrahydrofuran to give an anhydride, which was converted to N- in 80 m of anhydrous pyridine.
Aspirate through a frit through 14.35 g (73.5 mmol) of methylglucamine. Stir overnight, evaporate to dryness in vacuo,
The pyridine residue is removed by azeotropic distillation with ethanol,
Purify by chromatography on 800 g of silica gel using acetate / ethanol 2: 1 as eluent. Thus, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- [N- (2,
16.11 g (63.7% of theory) of 3,4,5,6-pentahydroxyhexyl) -N-methyl] -amide are obtained as an orange syrupy product.

C ₁₆ H ₂₉ N ₂ O ₇ (361.42) Calculated value: 53.17C 8.09H 7.75N Measured value: 52.93C 8.22H 7.58N Example 22 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-Carboxylic acid- (1,3-dihydroxyprop-2-yl) -amide As in Example 4, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid 5.50 g (29.86
Mmol), 3.27 g (32 mmol) of triethylamine, 3.58 g (32 mmol) of ethyl chloroformate and 2.92 of 2-amino-1,3-propanediol.
g (32 mmol) of anhydrous tetrahydrofuran 100 m
React inside. In this way, it will crystallize when left unattended
2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid having a melting point of 1 to 153 ° C.
(1,3-Dihydroxyprop-2-yl) -amide 5.
27 g (68.6% of theory) are obtained.

C₁₂H_{twenty one}N₂O_Four (257.31) Calculated: 56.01C 8.23H 10.89N Found: 55.80C 8.49H 10.71N Example 23 2,2,5,5-tetramethylpyrrolidine-1-oxy
Le-3-carboxylic acid-N- (1,3-dihydroxypro
P-2-yl) -amide 2,2,5,5-tetramethylpyrrolidin-1-oxy
Le-3-carboxylic acid methyl ester 1.00 g (5 mm
) And 2-amino-1,3-propanediol 0.68
g (7.5 mmol) for 6 h with argon.
Heat to 120 ° C under. After cooling, take in distilled water and
Extraction with ethyl ether and ion exchange (Amberlite I
R120H⁺Form) to bond excess amine
To do. The exchanger is filtered off, washed with water and the combined aqueous phases are vacuumed.
Evaporate to dryness in. Thus 2,2,5,5-tetra
Methylpyrrolidine-1-oxyl-3-carboxylic acid-
(1,3-Dihydroxyprop-2-yl) -amide 7
90 mg (60.8% of theory) as an orange oil
can get.

C ₁₂ H ₂₃ N ₂ O ₄ (259.33) Calculated: 55.58C 8.94H 10.80N Found: 55.29C 9.22H 10.72N Example 24 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- 3-Carboxylic acid (5-hydroxy-2,2-dimethyl-1,3-dioxepan-6-yl) -amide 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol in 100 m of dichloromethane 1.61g (1
0 mmol) in a solution of 2,2,5,5-tetramethyl-3-in 20 m of dichloromethane with stirring at 0 ° C.
2.563 g of a mixed anhydride consisting of pyrroline-1-oxyl-3-carboxylic acid and chloroformic acid ethyl ester (10
Solution) is added dropwise. After stirring for 60 minutes, the solution is washed with saturated sodium bicarbonate, the organic phase is separated off, dried over sodium sulphate and concentrated by evaporation in a vacuum. The residue is crystallized from diethyl ether / hexane. Thus, 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid having a melting point of 132 to 133 ° C.
(5-Hydroxy-2,2-dimethyl-1,3-dioxepan-6-yl) -amide 2.27 g (69.3% of theory)
Is obtained.

Example 25 Succinic acid- {N- [2-hydroxy-2- (2,2-dimethyl-1,3-dioxolan-4-yl) -ethyl]
-N '-(2,2,6,6-tetramethyl-1-oxylpiperidin-4-yl)} diamide succinic acid in 200 m of anhydrous tetrahydrofuran-
(2,2,6,6-tetramethyl-1-oxide-4-
To a solution of 2.71 g (10 mmol) of piperidyl) monoamide was added triethylamine 1. under stirring, cooling and nitrogen flow.
01 g (= 10 mmol) are added. Cool to -5 ° C, 2
During 0, 1.09 g (= 10 mmol) of methyl chloroformate dissolved in 10 m of anhydrous tetrahydrofuran
Is added dropwise with stirring. Stir for 30 minutes at -5 ° C.
Then, 2-amino-1- (2,2-dimethyl-1,3
-Dioxolan-4-yl) -ethanol 1.61 g (= 1
0 mmol) is added in portions. After 30 minutes, remove the cooling and stir at room temperature for 3 hours. Then, anhydrous ether 20
Dilute with 0 m, filter the solid with suction, wash with anhydrous ether and discard. The organic solution is evaporated to dryness in vacuo, the residue is taken up in dichloromethane, washed with a little ice-cold semisaturated sodium bicarbonate solution and dried over sodium sulphate, then the organic phase is evaporated to dryness again in vacuum. .
2.6 g (63% of theory) of an orange syrupy product are obtained.

C₂₀H₃₆N₃O₆ (414.520) Calculated: 57.95C 8.75H 10.14N Measured: 57.86C 9.00H 10.20N Example 26 Succinic acid- [N- (2,2,6,6-tetramethyl-1
-Oxylpiperidin-4-yl) -N '-(2,3,
4-trihydroxybutyl)] diamide In 50 m of water containing 0.1 m of concentrated sulfuric acid,
Curic acid-{-[2-hydroxy-2- (2,2-dimethyl
-1,3-Dioxolan-4-yl) -ethyl] -N '
-(2,2,6,6-tetramethyl-1-oxylpipe
Lysin-4-yl)} diamide 2.05 g (= 5 mmol)
To suspend. Heat to 50 ° C for 3 hours with stirring
It Then, it is cooled to room temperature and the solution is exchanged with an ion exchanger (Ambe
rlite IRA410 (OH^-Form) to neutralize. Filter the exchange
And the solution is evaporated to dryness in vacuo. Orange white
1.67 g (= 90% of theory) of lumps are obtained.

C ₁₇ H ₃₂ N ₃ O ₆ (374.456) Calculated value: 54.53C 8.61H 11.22N Measured value: 54.44C 8.80H 11.02N Example 27 2,2,6,6-tetramethyl-1,2,5,6- Tetrahydropyridine-1-oxyl-4-carboxylic acid-
[N- (2,3,4,5,6-pentahydroxyhexyl) -N-methyl] -amide As in Example 4, 2,2,6,6-tetramethyl-1,
2,5,6-tetrahydropyridine-1-oxyl-4
-7.93 g (40 mmol) of carboxylic acid, 4.3 g (42.5 mmol) of triethylamine, ethyl chloroformate
4.61 g (42.5 mmol) of anhydrous tetrahydrofuran 1
In 50m anhydrous pyridine to give 8.3g of N-methylglucamine (42.5m).
Through a fritted flask. Stir overnight, evaporate to concentration in vacuo, remove the pyridine residue by azeotropic distillation with ethanol and purify by chromatography on 500 g silica gel using acetate / ethanol 2: 1 as eluent. . Thus, 2, 2,
6,6-Tetramethyl-1,2,5,6-tetrahydropyridine-1-oxyl-4-carboxylic acid- [N-
(2,3,4,5,6-pentahydroxyhexyl)-
9.19 g (61.2% of theory) of N-methyl] -amide are obtained as an orange syrupy product.

C ₁₇ H ₃₁ N ₂ O ₇ (375.45) Calculated: 54.39 C 8.32H 7.46N Found: 54.12C 8.51H 7.30N Example 28 (1-oxyl-2,2,6,6-tetramethyl-piperidine-4) -Yl) -acetic acid- [N- (2,3,4,5,
6-Pentahydroxyhexyl) -N-methyl] -amide Similar to Example 4, (1-oxyl-2,2,6,6-tetramethyl-piperidin-4-yl) -acetic acid 8.57 g (40
Mmol), 4.3 g (42.5 mmol) of triethylamine and 4.61 g (4.25 mmol) of ethyl chloroformate are reacted in 100 m of anhydrous tetrahydrofuran to give a mixed anhydride, which is 8.3 g of N-methylglucamine in 50 m of anhydrous pyridine. Suction through the frit to (42.5 mmol). Work-up as described in Example 25 and thus (1-oxyl-2,2,6,6
-Tetramethylpiperidin-4-yl) -acetic acid- [N-
(2,3,4,5,6-pentahydroxyhexyl)-
9.36 g (59.8% of theory) of N-methyl] -amide are obtained as an orange syrupy product.

_{C 18 H 35 N 2 O 7} (391.49) Calculated: 55.23C 9.01H 7.16N Found: 55.44C 9.18H 7.02N Example 29 succinic acid - [N-(1-oxyl-2,2,5,5 Tetramethylpyrrolidin-3-yl) -N'-methyl-
N '-(2,3,4,5,6-pentahydroxyhexyl)]-diamide Similar to Example 4, succinic acid- (1-oxyl-2,2,2
5,5-Tetramethylpyrrolidin-3-yl) -monoamide 10.29 g (40 mmol) and triethylamine 4.3 g
(42.5 mmol) and 4.61 g of ethyl chloroformate
(42.5 mmol) and anhydrous tetrahydrofuran 100 m
In an anhydrous form, which is dried over 50 m of anhydrous pyridine.
Suction is added through a frit to 8.3 g (42.5 mmol) of N-methylglucamine in. Work-up as described in Example 25, thus giving succinic acid- [N- (1-
Oxyl-2,2,5,5-tetramethylpyrrolidine-
3-)-N'-methyl-N '-(2,3,4,5,6-
10.53 g (62.6% of theory) of pentahydroxyhexyl)]-diamide are obtained as an orange syrupy product.

C ₁₉ H ₃₆ N ₂ O ₈ (420.51) Calculated: 54.27C 8.63H 6.66N Found: 54.09C 8.47H 6.49N Example 30 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl- Solution of 3-carboxylic acid-2,3-dihydroxypropylamide 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid-2,3-dihydroxypropylamide 258.317 g (= 1 Mol) is dissolved in 600 m of water for injection while heating. Add 1.2 g of tromethamine, make up to 1000 m with water for injection and bottle the sterile filtered neutral solution.

NMR-Diagnostics Example 31 Production of Nitroxyl Loaded Liposomes Proc. Nat. Acad. Sci. 7th, USA
A resin mixture consisting of 50 parts egg-phosphatidylcholine and 50 parts cholesterol is prepared as a dry substance by the procedure described in Volume 5, page 4194. Of this, 500 mg was dissolved in 30 m of peroxide-free diethyl ether and the solution in water for injection was added in an ultrasonic bath.
2,5,5-tetramethylpyrrolidine-1-oxyl-
3-carboxylic acid- (2,3-dihydroxypropyl)-
3m of a 1 molar amide solution are added dropwise. After the addition is complete, continue to sonicate for a further 5 minutes, then concentrate by evaporation in a rotary evaporator. The microemulsion obtained is diluted with physiological saline and centrifuged at 2000C (20,000 g / 20 min) repeatedly to remove the unencapsulated control agent. After that, the liposomes
Lyophilize in l). The application is carried out as a dispersion in saline, which is 2.5-5.0 m when injected intravenously.
A dose of / kg evokes a good control delineating effect on the liver and spleen.

Example 32 Preparation of a solution of nitroxyl-protein-complex 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid 77.1 mg (= 0.5 mmol)
Is dissolved in 10 m of dimethyl sulfoxide. 2.0 m of dimethyl sulfoxide while cooling the solution and stirring
57.6 mg of N-hydroxysuccinimide (0.
Solution of dicyclohexylcarbodiimide 9 in 2.5 m of dimethyl sulfoxide.
A solution of 2.9 mg (0.45 mmol) is added dropwise. After stirring overnight, filtration is carried out, and an appropriate portion of this solution is gradually added to a desired molar ratio of an immunoglobulin G solution (immunoglobulin G solution) in 0.1 molar sodium phosphate / sodium chloride buffer (pH 8.0). G concentration: 2.6-3.4 mg / m). Stir at room temperature for 2 hours,
Separation by passing through a Bio-Gel P30 column,
Sequentially at 4 ° C for 24 hours 0.9% sodium chloride-0.0
800m of 2% sodium azide solution and 800m of 0.1M sodium citrate buffer (pH
Dialyse against 5.0). Lyophilization and cold storage give a stable dry formulation, which is dissolved in water for injection as needed.

Example 33 Complex consisting of bovine serum albumin (BSA) and 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid 12.0 g (179.1 mol) of BSA was distilled at 0 ° C. Melt in 200m. Then IN caustic soda solution 2
Add 0 m and dilute with 200 m of tetrahydrofuran. 2,2,5,5-tetramethylpyrrolidine-1-
2.17 g (11.65 mmol) of oxyl-3-carboxylic acid, 1.774 m (12.68 m) of triethylamine
Mol) and isobutyl chloroformate 1.83 m
(12.68 mmol) to anhydrous tetrahydrofuran 1
Prepare a mixed anhydride solution at -10 ° C in 00m,
Add a small amount to the BSA-solution so as not to exceed a temperature of 2 ° C. After stirring for a further 2 hours at this temperature, the batch is then carefully evaporated to dryness in vacuo with a bath temperature of 15 ° C. After dilution to 300 m, the solution is ultrafiltered through a YM30-membrane (this operation is carried out at + 4 ° C). The solution is subsequently freeze-dried. 10.5 g of lyophilisate is obtained as a friable white powder. Amino group measurement by Habeeb corresponds to 95.1% of theory.
The arrangement of the 49.5 amino group of the protein molecule was revealed.

This complex showed a surprisingly high stability of the nitroxyl group in the ascorbic acid test.

[Brief description of drawings]

FIG. 1 is a nuclear spin tomographic photograph of a tissue of a kidney portion having a colon cancer tumor of a mouse before the application of the contrast agent of the present invention, FIG. Fig. 4 is a photograph of the same structure after a minute, and Fig. 4 is a photograph collectively showing Fig. 1, Fig. 2, and Fig. 3 so that changes in the same structure can be observed with time.

Claims (33)

[Claims] 1. The general formula I ': [Wherein B is a protein residue, a lipid residue or a group- Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R
₃ and R ₄ represent an alkyl group, R ₅ and R ₆ represent an alkyl group optionally substituted by a hydroxy group, Is a double bond, m and n are zero, and R ₃ , R
₄ , R ₅ and R ₆ each represent a methyl group, and R ₂
Is a hydrogen atom, R ₁ is a 2,3-dihydroxy-prop-2-yl group, a 2,3,4-trihydroxybutyl group, a 1,3,4-trihydroxybut-2-yl group, Trishydroxymethylmethane group, 2,3,4,5
-Tetrahydroxypentyl group, 1,2,4,5-tetrahydroxypent-3-yl group, 2,3,4,5,6
-Pentahydroxyhexyl group or 1,3,4,5,
Represents a 6-pentahydroxyhex-2-yl group]. 2. 2,2,5,5-Tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- [2-hydroxy-
2- (2,2-dimethyl-1,3-dioxolane-4-
Ill) -ethyl] -amide.
A nitroxyl compound according to the item. 3. The method according to claim 1, which is 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (1,3-dihydroxyprop-2-yl) -amide. The described nitroxyl compound. 4. A 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylic acid- (2,2-dimethyl-1,3-dioxolan-4-yl-methyl) -amide. The nitroxyl compound according to claim 1. 5. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- [N- (2,2
A nitroxyl compound according to claim 1, which is -dimethyl-1,3-dioxolan-4-yl-methyl) -N-methyl] -amide. 6. The method according to claim 1, which is 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (2,3,4-trihydroxybutyl) -amide. Nitroxyl compounds. 7. 2,2,5,5-Tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- [N- (2,3,
The nitroxyl compound according to claim 1, which is 4,5,6-pentahydroxyhexyl) -N-methyl] -amide. 8. The method according to claim 1, which is 2,2,5,5-tetramethyl-pyrrolidine-1-oxyl-3-carboxylic acid- (2,3-dihydroxypropyl) -amide.
A nitroxyl compound according to the item. 9. Nitroxyl according to claim 1, which is 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid-bis- (2-hydroxyhexylethyl) -amide. Compound. 10. 2,2,5,5-Tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- [N- (2,2-
Dimethyl-1,3-dioxolan-4-yl-methyl)
The nitroxyl compound according to claim 1, which is -N-methyl] -amide. 11. A compound of claim 2, which is 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid-N- (2,3-dihydroxypropyl) -N-methyl-amide. A nitroxyl compound according to item 1. 12. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-carboxylic acid- [2-hydroxy-2- (2,2-dimethyl-1,3-dioxolane-4]
A nitroxyl compound according to claim 1, which is -yl) -ethyl] -amide. 13. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (2,3,4
A nitroxyl compound according to claim 1, which is -trihydroxybutyl) -amide. 14. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (5-hydroxy-2,2-dimethyl-1,3-dioxepane-6-
The nitroxyl compound according to claim 1, which is yl) -amide. 15. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (1,3,4
A nitroxyl compound according to claim 1, which is -trihydroxybut-2-yl) -amide. 16. 2,2,5,5-Tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (5-hydroxy-2,2-dimethyl-1,3-dioxepan-6-yl) -amide A nitroxyl compound according to claim 1. 17. A 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid- (1,3,4-trihydroxybut-2-yl) -amide which is a claim. The nitroxyl compound according to item 1. 18. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- [N- (2,
The nitroxyl compound according to claim 1, which is 3,4,5,6-pentahydroxyhexyl) -N-methyl] -amide. 19. A 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (1,3-dihydroxyprop-2-yl) -amide. The nitroxyl compound according to item 1. 20. 2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (5-hydroxy-2,2-dimethyl-1,3-dioxepane-6-
The nitroxyl compound according to claim 1, which is yl) -amide. 21. Succinic acid-N- [2-hydroxy-2-
(2,2-Dimethyl-1,3-dioxolan-4-yl) -ethyl] -N '(2,2,6,6-tetramethyl-1-oxyl-piperidin-4-yl) -diamide. The nitroxyl compound according to claim 1. 22. Succinic acid- [N- (2,2,6,6-tetramethyl-1-oxyl-piperidin-4-yl)-
The nitroxyl compound according to claim 1, which is N '-(2,3,4-trihydroxybutyl)]-diamide. 23. (1-Oxyl-2,2,6,6-tetramethyl-piperidin-4-yl) -acetic acid- [N-
(2,3,4,5,6-pentahydroxyhexyl)-
The nitroxyl compound according to claim 1, which is N-methyl] -amide. 24. Succinic acid- [N- (1-oxyl-2,
2,5,5-Tetramethylpyrrolidin-3-yl)-
The nitroxyl compound according to claim 1, which is N'-methyl-N '(2,3,4,5,6-pentahydroxyhexyl)]-diamide. 25. The general formula I ′ [Wherein B is a group Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R
₃ and R ₄ represent an alkyl group, R ₅ and R ₆ represent an alkyl group optionally substituted by a hydroxy group, Is a double bond, m and n are zero, and R ₃ , R
₄ , R ₅ and R ₆ each represent a methyl group, and R ₂
Is a hydrogen atom, R ₁ is a 2,3-dihydroxy-prop-2-yl group, a 2,3,4-trihydroxybutyl group, a 1,3,4-trihydroxybut-2-yl group, Trishydroxymethylmethane group, 2,3,4,5
-Tetrahydroxypentyl group, 1,2,4,5-tetrahydroxypent-3-yl group, 2,3,4,5,6
-Pentahydroxyhexyl group or 1,3,4,5,
In the method for producing a nitroxyl compound represented by the formula: 6-pentahydroxyhex-2-yl group]
I: [In the formula m, X, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are the same as those described above, and Y is a hydrogen atom or a hydroxy group, and a ketal group optionally present is oxidized. Alternatively, a method for producing a novel nitroxyl compound, which comprises hydrolyzing and eliminating an acyl group. 26. The general formula I ′ [Wherein B is a group Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R
₃ and R ₄ represent an alkyl group, R ₅ and R ₆ represent an alkyl group optionally substituted by a hydroxy group, Is a double bond, m and n are zero, and R ₃ , R
₄ , R ₅ and R ₆ each represent a methyl group, and R ₂
Is a hydrogen atom, R ₁ is a 2,3-dihydroxy-prop-2-yl group, a 2,3,4-trihydroxybutyl group, a 1,3,4-trihydroxybut-2-yl group, Trishydroxymethylmethane group, 2,3,4,5
-Tetrahydroxypentyl group, 1,2,4,5-tetrahydroxypent-3-yl group, 2,3,4,5,6
-Pentahydroxyhexyl group or 1,3,4,5,
In the process for producing a nitroxyl compound represented by 6-pentahydroxyhex-2-yl group]
I: [In the formula m, X, R ₃ , R ₄ , R ₅ and R ₆ are as defined above] or a reactive derivative of this carboxylic acid with the general formula IV: A process for producing a novel nitroxyl compound, which comprises condensing with an amine represented by the formula [wherein R ₁ and R ₂ represent the same as those defined above] and hydrolyzing and eliminating an optional ketal group or acyl group. 27. General formula I ′: [Wherein B is a group Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R
₃ and R ₄ represent an alkyl group, R ₅ and R ₆ represent an alkyl group optionally substituted by a hydroxy group, Is a double bond, m and n are zero, and R ₃ , R
₄ , R ₅ and R ₆ each represent a methyl group, and R ₂
Is a hydrogen atom, R ₁ is a 2,3-dihydroxy-prop-2-yl group, a 2,3,4-trihydroxybutyl group, a 1,3,4-trihydroxybut-2-yl group, Trishydroxymethylmethane group, 2,3,4,5
-Tetrahydroxypentyl group, 1,2,4,5-tetrahydroxypent-3-yl group, 2,3,4,5,6
-Pentahydroxyhexyl group or 1,3,4,5,
In the process for producing a nitroxyl compound represented by 6-pentahydroxyhex-2-yl group], the compound represented by the general formula V: A carboxylic acid represented by the formula [wherein n, R ₁ and R ₂ are the same as those defined above] or a reactive derivative of the carboxylic acid represented by the general formula VI: [Wherein m, R ₃ , R ₄ , R ₅ and R ₆ are the same as those defined above], and a ketal group or an acyl group optionally present is hydrolyzed and eliminated. A method for producing a novel nitroxyl compound. 28. General formula I ′: [Wherein B represents a protein residue or a lipid residue, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, and R ₂ represents the same as R ₁ or a hydrogen atom. Or represents an alkyl group,
R ₃ and R ₄ represent an alkyl group, and R ₅ and R ₆
Represents an alkyl group optionally substituted by a hydroxy group, Is a double bond, m and n are zero, and R ₃ , R
₄ , R ₅ and R ₆ each represent a methyl group, and R ₂
Is a hydrogen atom, R ₁ is a 2,3-dihydroxy-prop-2-yl group, a 2,3,4-trihydroxybutyl group, a 1,3,4-trihydroxybut-2-yl group, Trishydroxymethylmethane group, 2,3,4,5
-Tetrahydroxypentyl group, 1,2,4,5-tetrahydroxypent-3-yl group, 2,3,4,5,6
-Pentahydroxyhexyl group or 1,3,4,5,
In the process for producing a nitroxyl compound represented by 6-pentahydroxyhex-2-yl group]
I: [In the formula m, X, R ₃ , R ₄ , R ₅ or R ₆ represents the above-mentioned], or a reactive derivative of this carboxylic acid with a nucleophilic group of a protein or lipid. A method for producing a novel nitroxyl compound characterized by 29. General formula I: [Wherein B is a protein residue, a lipid residue or a group- Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R ₃
And R ₄ represents an alkyl group, and R ₅ and R
₆ represents an alkyl group optionally substituted by a hydroxy group], and an NMR diagnostic agent is contained. 30. An NM according to claim 29, which contains a compound of the general formula (I) in which B represents a protein residue.
R diagnostic agent. 31. A composition according to claim 2, which contains 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (2-hydroxyethyl) -amide.
Item 9. The NMR diagnostic agent according to Item 9. 32. The method according to claim 29, which contains 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid- (2,3-dihydroxypropyl) -amide. The NMR diagnostic agent described. 33. General formula I: [Wherein B is a protein residue, a lipid residue or a group- Represents, Represents a single bond or a double bond, X is a group - (CH _{2) n} - or represents, or Group -NHCO but if a single bond (CH _{2) n} - (where n = 0
To 4), m represents a number of 0 or 1, R ₁ represents an alkyl group substituted by a hydroxy group or an alkylidenedioxy group, R ₂ represents the same as R ₁ or a hydrogen atom or Represents an alkyl group, R ₃
And R ₄ represents an alkyl group, and R ₅ and R
₆ represents an alkyl group optionally substituted by a hydroxy group] as an inclusion compound in a ribosome, an NMR diagnostic agent.